The ADP-ribosylation factors (Arfs) are a family of small GTPases that regulate vesicular transport in all eukaryotic cells. The primary role of Arfs in this context is to nucleate the assembly of coat protein complexes at sites of carrier vesicle formation. These coat complexes select and concentrate cargo for transport, and also provide the energy to form a vesicle. It is well established that Arfs promote the assembly of clathrin-coated vesicles in the Golgi apparatus and on endosomes, but their role in clathrin- mediated endocytosis at the plasma membrane is poorly understood. Of the six mammalian Arf isoforms, Arf6 is most abundant in the cell periphery, where it has been shown to regulate the clathrin-dependent endocytosis of G-protein coupled receptors, but not of transferrin receptor, whose internalization is also clathrin dependent. We hypothesize that Arf6 regulates the endocytosis of a subset of plasma membrane proteins that require the participation of monomeric adaptor proteins to link them to the clathrin endocytic machinery. In Aim 1, we will examine the role of Arf6 in the endocytosis and postendocytic transport of a panel of membrane proteins that exhibit different modes of interaction with the endocytic machinery. Arf6 also functions on endosomes to regulate postendocytic recycling, and we hypothesize that its activation at each location is differentially regulated by site-specific guanine nucleotide exchange factors (GEFs). Aim 1 will also explore the role of different Arf6 GEFs in endocytic and postendocytic transport. We have begun to characterize a class of Arf6-specific GEFs, the BRAGs (Brefeldin Resistant Arf GEFs) that bind both clathrin and the AP-2 adaptor complex. In Aim 2, we will define the specific functions of the three BRAG isoforms in endocytic processes, and perform a structure/function analysis to define their mechanisms of action. Finally, our preliminary data indicate that Arf6 binds to a family of monomeric adaptor proteins containing N-terminal phosphotyrosine binding domains (PTBs) that have been shown to link proteins containing NPXY sorting signals to the clathrin endocytic machinery. In Aim 3 we will determine how Arf6 may regulate interaction of these proteins with cargo and the clathrin/AP-2 complex. Because this class of cargo molecules includes key regulators of cholesterol homeostasis, cell adhesion and neuronal function, the proposed studies will provide fundamental insights into the mechanisms by which these processes are regulated.